The aims of this study were to assess the renal expression of angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and MAS receptor in human type 2 diabetic nephropathy (DN).

The human pathology Wilson disease (WD) is characterized by toxic copper (Cu) accumulation in brain and liver, resulting in, among other indications, mitochondrial dysfunction and apoptosis of hepatocytes. In an effort to identify novel compounds that can alleviate Cu-induced toxicity, we screened the Pharmakon 1600 repositioning library using a Cu-toxicity yeast screen. We identified 2 members of the drug class of Angiotensin II Type 1 receptor blockers (ARBs) that could increase yeast tolerance to Cu, namely Candesartan and Losartan. Subsequently, we show that specific ARBs can increase yeast tolerance to Cu and/or the chemotherapeutic agent cisplatin (Cp). The latter also induces mitochondrial dysfunction and apoptosis in mammalian cells. We further demonstrate that specific ARBs can prevent the prevalence of Cu-induced apoptotic markers in yeast, with Candesartan Cilexetil being the ARB which demonstrated most pronounced reduction of apoptosis-related markers. Next, we tested the sensitivity of a selection of yeast knockout mutants affected in detoxification of reactive oxygen species (ROS) and Cu for Candesartan Cilexetil rescue in presence of Cu. These data indicate that Candesartan Cilexetil increases yeast tolerance to Cu irrespectively of major ROS-detoxifying proteins. Finally, we show that specific ARBs can increase mammalian cell tolerance to Cu, as well as decrease the prevalence of Cu-induced apoptotic markers. All the above point to the potential of ARBs in preventing Cu-induced toxicity in yeast and mammalian cells.

Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensive drugs. Apart from their activity towards the circulatory system, ARBs also penetrate the blood-brain barrier and display neuroprotective effects. Kynurenic acid (KYNA) is an endogenous metabolite of tryptophan produced by kynurenine aminotransferase II (KAT II) in the brain. Antagonism towards all ionotropic glutamate (GLU) receptors is the main mechanism of KYNA action. An elevated brain level of KYNA is linked with memory impairment and psychotic symptoms. The aim of this study was to examine the influence of three ARBs: irbesartan, losartan, and telmisartan on KYNA production and KAT II activity in rat brain. The effect of ARBs on KYNA production was analyzed in rat brain cortical slices and on isolated KAT II enzyme. Irbesartan, losartan, and telmisartan decreased KYNA production and KAT II activity in a dose-dependent manner in rat brain cortex in vitro. Molecular docking suggested that the examined ARBs could bind to an active site of KAT II. In conclusion, ARBs decrease KYNA production in rat brain by direct inhibition of KAT II enzymatic activity. This novel mechanism of ARBs action may be advantageous in the treatment of cognitive impairment or the management of schizophrenia.

While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr(113), Tyr(184), Lys(199), His(256) and Gln(257) using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr(113) in the AT1 receptor and the hydroxyl group of olmesartan, between Lys(199) and carboxyl or tetrazole groups, and between His(256) or Gln(257) and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys(199), His(256) and Gln(257) of AT1 receptor. Lys(199) in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys(199). The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr(113). On the other hand, the n-butyl group of irbesartan may bind to Tyr(113). In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (termed ventilator-induced diaphragm dysfunction (VIDD)). Although VIDD is a major risk factor for problems in weaning patients from MV, a standard therapy to prevent VIDD does not exist. However, emerging evidence suggests that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the essential characteristics of AT1R blockers (ARBs) required to protect against VIDD remain unclear. To determine the traits of ARBs that are vital for protection against VIDD, we compared the efficacy of two clinically relevant ARBs, irbesartan and olmesartan; these ARBs differ in molecular structure and effects on AT1Rs. Specifically, olmesartan blocks both angiotensin II (AngII) binding and mechanical activation of AT1Rs, whereas irbesartan prevents only AngII binding to AT1Rs. Using a well-established preclinical model of prolonged MV, we tested the hypothesis that compared with irbesartan, olmesartan provides greater protection against VIDD. Our results reveal that irbesartan does not protect against VIDD whereas olmesartan defends against both MV-induced diaphragmatic atrophy and contractile dysfunction. These findings support the hypothesis that olmesartan is superior to irbesartan in protecting against VIDD and are consistent with the concept that blockade of mechanical activation of AT1Rs is a required property of ARBs to shield against VIDD. These important findings provide a foundation for future clinical trials to evaluate ARBs as a therapy to protect against VIDD.

Angiotensin II type 1 receptor antibody (AT1R-Ab), is a non-human leukocyte antigen (HLA) antibody implicated in poor renal allograft outcomes, although its actions may be mediated through a different pathway than HLA donor-specific antibodies (DSAs). Our aim was to examine serum cytokine profiles associated with AT1R-Ab and distinguish them from those associated with HLA DSA in serially collected blood samples from a cohort of pediatric renal transplant recipients.

Angiotensin II type 1 receptor blockers (ARBs) have been investigated for their neuroprotective and intraocular pressure (IOP) lowering effects in treating glaucoma, but the reports have been inconsistent possibly because different compounds and models have been used. Here we selected three ARBs for head-to-head comparisons of their effects on IOP and transforming growth factor β (TGFβ) signaling, which is believed to play an important role in glaucoma pathogenesis.

Background Angiotensin II type 1 receptor blockers (ARBs) have been shown to limit the growth of abdominal aortic aneurysm (AAA), but their efficacy is controversial. This study aimed to investigate the molecular mechanism underlying the protective effect of ARBs against AAA progression. Methods and Results Olmesartan, an ARB, was administered to wild-type and osteoprotegerin-knockout (Opg-KO) mice starting 2 weeks before direct application of CaCl2 to aortas to induce AAA. The protective effect of olmesartan against AAA in wild-type and Opg-KO mice was compared at 6 weeks after AAA induction. Olmesartan prevented AAA progression in Opg-KO mice, including excessive aortic dilatation and collapse of tunica media, but not in wild-type mice. Deficiency of the Opg gene is known to cause excessive activation of the tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase/matrix metalloproteinase 9 pathway, resulting in prolonged AAA progression. Olmesartan attenuated the upregulation of phosphorylated c-Jun N-terminal kinase and matrix metalloproteinase 9 expression in the aortic wall of Opg-KO mice. In cultured vascular smooth muscle cells, tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase phosphorylation and matrix metalloproteinase 9 expression were inhibited by angiotensin (1-7), the circulating levels of which are increased by ARBs. Furthermore, administering an angiotensin (1-7) antagonist to Opg-KO mice diminished the protective effect of olmesartan against AAA progression. Conclusions Olmesartan prevented AAA progression in Opg-KO mice by upregulating angiotensin (1-7), suggesting that angiotensin (1-7) may be a key factor that mediates the protective effect of ARBs.

Previously, we demonstrated increased Angiotensin II type I receptor expression in leukocytes from patients with untreated, but not in treated, essential hypertension (essential hypertension). We hypothesized that the Angiotensin II AT1 receptor is also increased in leukocytes from patients with chronic kidney disease, however and can still be corrected with combined anti-hypertensive treatment with renin-angiotensin system (RAS) blockers and statins. Blood pressure, cholesterol, renal function oxidative stress parameters, inflammation, and leukocyte Angiotensin II AT1 receptor mRNA expression were measured both on and (6 weeks) off treatment. Data were compared to data of 10 healthy control subjects. Untreated chronic kidney disease patients (n=20) had higher blood pressure, cholesterol and leukocyte Angiotensin II AT1 receptor mRNA expression, but no different ox-LDL, thiobarbituric acid reactive substances, paraoxonase activity or hs-CRP. OxLDL and Lipoprotein(a) were increased in untreated chronic kidney disease. Angiotensin II AT1 receptor expression inversely correlated with renal function (R(2)=0.15, P<0.03) and Lipoprotein(a) but not with the other parameters. Treatment with RAS blockers and statins normalized blood pressure and cholesterol, however it did not correct enhanced leukocyte Angiotensin II AT1 receptor expression. Leukocyte Angiotensin II AT1 receptor expression is inappropriately high in chronic kidney disease, correlates inversely with renal function and does not depend on antihypertensive and lipid-lowering treatment. The uremic environment seems to dominate over previously reported actions of high blood pressure and cholesterol to enhance leukocyte Angiotensin II AT1 receptor expression.

Gastric cancer with peritoneal dissemination has poor clinical prognosis because of the presence of rich stromal fibrosis and acquired drug resistance. Recently, Angiotensin II type I receptor blockers such as candesartan have attracted attention for their potential anti-fibrotic activity. We examined whether candesartan could attenuate tumor proliferation and fibrosis through the interaction between gastric cancer cell line (MKN45) cells and human peritoneal mesothelial cells. Candesartan significantly reduced TGF-β1 expression and epithelial-to-mesenchymal transition-like change, while tumor proliferation and stromal fibrosis were impaired. Targeting the Angiotensin II signaling pathway may therefore be an efficient strategy for treatment of tumor proliferation and fibrosis.

Several comorbidities, including hypertension, have been associated with an increased risk of developing severe disease during SARS-CoV-2 infection. Angiotensin II receptor blockers (ARBs) are currently some of the most widely-used drugs to control blood pressure by acting on the angiotensin II type 1 receptor (AT1R). ARBs have been reported to trigger the modulation of the angiotensin I converting enzyme 2 (ACE2), the receptor used by the virus to penetrate susceptible cells, raising concern that such treatments may promote virus capture and increase their viral load in patients receiving ARBs therapy. In this in vitro study, we reviewed the effect of ARBs on ACE2 and AT1R expression and investigated whether treatment of permissive ACE2+/AT1R+ Vero E6 cells with ARBs alters SARS-CoV-2 replication in vitro in an angiotensin II-free system. After treating the cells with the ARBs, we observed an approximate 50% relative increase in SARS-CoV-2 production in infected Vero E6 cells that correlates with the ARBs-induced up-regulation of ACE2 expression. From this data, we believe that the use of ARBs in hypertensive patients infected by SARS-CoV-2 should be carefully evaluated.

Blockers of G-protein coupled receptors (GPCRs), angiotensin II (Ang II) type 1 (AT1) receptor and β1-adrenergic (Ad) receptor, have been shown to improve the prognosis of cardiovascular disease. Cholesterol molecules in the cell membrane are needed to stabilize GPCRs as well as the cell membrane itself. We determined whether the functions of AT1 and β1-Ad receptors were changed by cholesterol depletion from cardiovascular cell membranes. Ang II-induced inositol phosphate production through AT1 receptor was suppressed by cholesterol depletion from cell membranes using rosuvastatin or methyl-β-cyclodextrin (MβCD), whereas isoproterenol-induced cyclic AMP production through β1-Ad receptor did not change after cholesterol depletion. In addition, the binding affinities of Ang II and AT1 receptor blocker after cholesterol depletion were significantly lower than those before depletion. Although AT1 receptor expression levels did not change after cholesterol depletion, the expression levels of AT1 receptor that could bind to Ang II significantly decreased after depletion. The changes in the structure of AT1 receptor due to depletion were confirmed by substituted-cysteine accessibility mapping. In conclusion, Ang II-induced activation of AT1 receptor is reduced without affecting the function of β1-Ad receptor after cholesterol depletion from cardiovascular cell membranes.

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

Staphylococcal nuclease domain containing-1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC) patients and promotes tumorigenesis by human HCC cells. We now document that SND1 increases angiotensin II type 1 receptor (AT1R) levels by increasing AT1R mRNA stability. This results in activation of ERK, Smad2 and subsequently the TGFβ signaling pathway, promoting epithelial-mesenchymal transition (EMT) and migration and invasion by human HCC cells. A positive correlation was observed between SND1 and AT1R expression levels in human HCC patients. Small molecule inhibitors of SND1, alone or in combination with AT1R blockers, might be an effective therapeutic strategy for late-stage aggressive HCC.

There are no therapeutics that directly enhance chronic endothelial nitric oxide (NO) release, which is typically associated with vascular homeostasis. In contrast, angiotensin II (AngII) receptor type 1 (AT1R) blockers (ARBs) can attenuate AngII-mediated oxidative stress, which often leads to increased endothelial NO bioavailability. Herein, we investigate the potential presence of direct, AngII/AT1R-independent ARB class effects on endothelial NO release and how this may result in enhanced aortic wall homeostasis and endothelial NO-specific transcriptome changes. Treatment of mice with four different ARBs induced sustained, long-term inhibition of vascular contractility by up to 82% at 16 weeks and 63% at 2 weeks, an effect reversed by L-NAME and absent in endothelial NO synthase (eNOS) KO mice or angiotensin converting enzyme inhibitor captopril-treated animals. In absence of AngII or in tissues with blunted AT1R expression or incubated with an AT2R blocker, telmisartan reduced vascular tone, supporting AngII/AT1R-independent pleiotropism. Finally, telmisartan was able to inhibit aging- and Marfan syndrome (MFS)-associated aortic root widening in NO-sensitive, BP-independent fashions, and correct aberrant TGF-β signaling. RNAseq analyses of aortic tissues identified early eNOS-specific transcriptome reprogramming of the aortic wall in response to telmisartan. This study suggests that ARBs are capable of major class effects on vasodilatory NO release in fashions that may not involve blockade of the AngII/AT1R pathway. Broader prophylactic use of ARBs along with identification of non-AngII/AT1R pathways activated by telmisartan should be investigated.

The renin angiotensin system (RAS) regulates numerous systemic functions and is expressed locally in skeletal tissues. Angiotensin1-7 (Ang1-7) is a beneficial member of the RAS, and the therapeutic effects of a large number of angiotensin receptors blockers (ARBs) are mediated by an Ang1-7-dependent cascade. This study examines whether the reported osteo-preservative effects of losartan are mediated through the angiotensin converting enzyme2 (ACE-2)/Ang1-7/Mas pathway in ovariectomized (OVX) rats. Sham and OVX animals received losartan (10mg/kg/d p.o.) for 6 weeks. A specific Mas receptor blocker (A-779) was delivered via mini-osmotic pumps during the losartan treatment period. Serum and urine bone metabolism biomarker levels were measured. Bone trabecular and cortical morphometry were quantified in distal femurs, whereas mineral contents were estimated in ashed bones, serum and urine. Finally, the expression of RAS components, the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was determined. Losartan significantly improved the elevated bone metabolism marker levels and altered trabecular and cortical structures in OVX animals, and restored normal urinary and skeletal mineral levels. Mas receptor inhibition significantly abolished all osteo-protective effects of losartan and enhanced the deleterious effects of OVX. Losartan enhanced OVX-induced up-regulation of ACE-1, AngII, angiotensin type 1 (AT1) receptor and RANKL expression, and increased ACE-2, Ang1-7, Mas and OPG expression in OVX animals. However, A-779 significantly eradicated the effects of losartan on RAS components and RANKL/OPG expression. Thus, Ang1-7 are involved in the osteo-preservative effects of losartan via Mas receptor, which may add therapeutic value to this well-known antihypertensive agent.

Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, β-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R/ETAR antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long-term treatment with receptor antibodies. Imaging studies revealed that β2- but not β1-arrestin was recruited to ETAR in response to ET-1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of β2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R- and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R/ETAR antibodies induce ERK1/2 and mTOR signaling involving β2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rationale for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

To assess the beneficial effects of the angiotensin II type 1 receptor blocker telmisartan on a non-obese animal model of reduced function and mass of islet beta-cells prior to the development of diabetes, Spontaneously Diabetic Torii (SDT) rats were treated with telmisartan at 8 weeks of age. At 24 weeks of age, the treatment with telmisartan dose-dependently ameliorated hyperglycemia and hypoinsulinemia, and high-dose (5 mg/kg/day) treated SDT rats did not developed diabetes. Real-time RT-PCR analysis revealed that treatment with high-dose telmisartan reduced mRNA expression of local renin-angiotensin system (RAS) components, components of NAD(P)H oxidase, transforming growth factor-beta1 and vascular endothelial growth factor in the pancreas of male SDT rats. Immunohistochemical and Western blot analyses revealed that treatment with telmisartan also reduced expression of p47(phox). These results suggest that treatment with telmisartan reduces oxidative stress by local RAS activation and protects against islet beta-cell damage and dysfunction. These findings provide at least a partial explanation for the reduced incidence of new-onset diabetes that has been observed in several clinical trials involving angiotensin II type 1 receptor blockers and ACE inhibitors.

Hypoxic tumour cells are radiation-resistant and are associated with poor therapeutic outcome. A poorly understood source of tumour hypoxia is unstable perfusion, which exposes tumour cells to varying oxygen tensions over time creating "transiently" hypoxic cells. Evidence suggests that angiotensin II type 1 receptor blockers (ARBs) can improve tumour perfusion by reducing collagen deposition from cancer associated fibroblasts (CAFs). However, the influence of ARBs on transient hypoxia and tumour radiation response is unknown. We tested how the ARBs losartan and telmisartan affected the solid tumour microenvironment, using fluorescent perfusion dyes and positron emission tomography to quantify tumour perfusion, and a combination of hypoxia markers and the hemorheological agent pentoxifylline to assess transient tumour hypoxia. We found CAF-containing tumours have reduced collagen I levels in response to telmisartan, but not losartan. Telmisartan significantly increased tumour blood flow, stabilized microregional tumour perfusion, and decreased tumour hypoxia by reducing the development of transient hypoxia. Telmisartan-treated tumours were more responsive to radiation, indicating that telmisartan reduces a therapeutically important population of transiently hypoxic tumour cells. Our findings indicate telmisartan is capable of modifying the tumour microenvironment to stabilize tumour perfusion, reduce transient hypoxia, and improve tumour radiation response.

Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal) nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP3) is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.